Facile Functionalization of Carbon Electrodes for Efficient Electroenzymatic Hydrogen Production

Enzymatic electrocatalysis holds promise for new biotechnological approaches to produce chemical commodities such as molecular hydrogen (H2). However, typical inhibitory limitations include low stability and/or low electrocatalytic currents (low product yields). Here we report a facile single-step electrode preparation procedure using indium–tin oxide nanoparticles on carbon electrodes. The subsequent immobilization of a model [FeFe]-hydrogenase from Clostridium pasteurianum (“CpI”) on the functionalized carbon electrode permits comparatively large quantities of H2 to be produced in a stable manner. Specifically, we observe current densities of >8 mA/cm2 at −0.8 V vs the standard hydrogen electrode (SHE) by direct electron transfer (DET) from cyclic voltammetry, with an onset potential for H2 production close to its standard potential at pH 7 (approximately −0.4 V vs. SHE). Importantly, hydrogenase-modified electrodes show high stability retaining ∼92% of their electrocatalytic current after 120 h of continuous potentiostatic H2 production at −0.6 V vs. SHE; gas chromatography confirmed ∼100% Faradaic efficiency. As the bioelectrode preparation method balances simplicity, performance, and stability, it paves the way for DET on other electroenzymatic reactions as well as semiartificial photosynthesis.


Contents
All subsequent steps were performed in the absence of dithionite to yield dithionite-free CpI.
[FeFe]-hydrogenase elution was performed with 50 mM biotin, which was subsequently removed with a desalting column (HiPrep 26/10 Desalting). The purified enzyme was concentrated to approximately 5 mg/mL and flash frozen in liquid N 2 . The specific activity of [FeFe]-hydrogenase was 135 ± 24 µmol H 2 min −1 mg −1 , as determined by online gas chromatography (GC). The specific activity was determined within an Ar atmosphere anoxic glovebox (Jacomex, France) at 30°C in MOPS buffer (0.1 M, pH 7) using 1 mM methyl viologen as the electron mediator and 100 mM dithionite as the overall electron donor.

S1.2 Preparation of nanoITO functionalized electrode
The preparation of a stock nanoITO colloidal suspension was modified from the method developed by Meyer and co-workers. S3 A 20% by weight indium tin oxide nanoparticle (nanoITO, In 2 O 3 :SnO 2 9:1 wt%, 17-28 nm APS, 99.5%, Thermo Scientific) suspension was prepared in a solution of 5 M acetic acid in absolute ethanol and sonicated for 20 min to ensure homogeneity. The volume of nanoITO suspension drop coated on the electrode was optimized for a given electrode surface area, as shown in Table S1. Note that a good nanoITO film should uniformly cover the whole electrode active area and be pinhole-free (Figure 1a).
After the evaporation of solvent (around 1 min), nanoITO coated working electrodes were annealed at 80°C in air for 20 min to achieve improved contact. Note that in Meyer's procedures, S3 the nanoITO electrodes were annealed at 500°C for 1 hour and then at 300°C under 3% H 2 /N 2 for 1 hour, which is modified to 80°C in air for 20 min to better adapt carbon electrodes.  Cyclic voltammetry of GCE-nanoITO-hydrogenase electrodes (third scan, scan rate: 10 mV/s) with (a) different nanoITO volume with 5 µL hydrogenase and (b) different hydrogenase volume with 1.5 µL nanoITO. Note that 3 mm GCE electrode cannot hold more than 1.5 µL nanoITO.

S3 Atomic force microscopy (AFM)
Images of the nanoITO electrode were acquired in amplitude modulated mode (AM-AFM) with an Infinity MFP3D AFM (Asylum Research, Oxford Instruments). AC240TS cantilevers (Olympus, Japan) were excited close to their resonance frequencies ( 70 kHz) and with free oscillation amplitudes (FOA) of approximately 160 nm. The set point and the scan rate were around 50% of the FOA and 0.2 Hz respectively. A typical image of a freshly prepared electrode is shown in Figure S2a. Section profiles along the black and red lines are shown in Figure S2b. Section profiles along lines 1 (black) and 2 (red).

S8
To determine the thickness of the nanoparticle layer, a freshly prepared nanoITO electrode was scratched with a knife edge to produce a small groove on the surface. Subsequently, an AFM topography is recorded in vicinity of the boundary of the scratch. In Figure S3a the topographic image of such area and in Figure S3b

S4 Repeats of hydrogenase electrochemistry
To validate the reproducibility of our reported hydrogenase electrochemistry. We performed CV on 5 different GCE-nanoITO-hydrogenase electrodes in Figure S4a and 2 different PGE-nanoITO-hydrogenase electrodes in Figure S4b. Figure S4c,d show 5 consecutive CV scans on 2 GCE electrodes where all CV traces are reasonably overlapped, confirming the electrochemical properties of the electrodes did not change.

S6 Hydrogenase electrochemistry without nanoITO
To validate the catalytic current density enhancement is not only as result of surface roughness, hydrogenase electrochemistry has been performed on bare GCE ( Figure S6a) and bare PGE ( Figure S6c). As a comparison, corresponding nanoITO modified electrodes have been plotted in Figure S6b

S7 O 2 -deactivation control experiments
To validate the catalytic current is originated from [FeFe]-hydrogenase instead of nanoITO. A O 2 -deactivation control experiment was performed by leaving the GCE-nanoITO-hydrogenase electrode under ambient condition for 20 min. The high oxygen level (around 21%) and the drying process were mean to deactivate the [FeFe]-hydrogenase. As we observed in Figure S7, deactivated bioelectrode shows a significantly lower current density than that of functional bioelectrode, around 17% at −0.8 V vs. SHE.

S8 H 2 oxidation experiments
A 3 mm GCE nanoITO-hydrogenase electrode was prepared as described in Section S1.2 and dropcast with 5 µL CpI. A gastight electrochemical cell was assembled under Ar, as described in Section S12. H 2 gas was introduced through a needle inserted into the cell septum and gently bubbled into the buffer for 3 min, then vented to ambient pressure. A CV was recorded (conditions in Section S1.3) before and after H 2 addition.    Figure S9: Additional stability tests for 1.5 h, 120.5 h, and 280.5 h. Corresponding 3rd CV scans before and after 280.5 h test. S17 S11 Bradford protein assay for hydrogenase leaching To quantify the amount of hydrogenase leaching from the nanoITO electrode in the solution as a function of time, a micro-Bradford protein assay was performed. A nanoITO electrode was functionalized with hydrogenase (7.05 mg/ml) and dried under ambient conditions for 9 min. The functionalized electrode was immersed in buffer (stirred) and an aliquot of the solution was taken at 2, 10, and 30 min.
A bovine serum albumin (BSA) protein standard (200 mg/ml, Sigma-Aldrich) was used to create an adjusted calibration range between 1 and 5 µg/ml, while a Bradford dye (ITW Reagents, Switzerland) was used for protein staining. A 96 well plate was used for samples (1:1 ratio sample:dye) and shaken for 5 min in a microplate reader (ThermoScientific Multiskan FC), before the absorbance was recorded at 595 nm.

Buffer
Stirrer 3 mm GCE-nanoITO 5 µL hydrogenase, dried 9 min To GC Column Figure S11: Sketch of the experimental setup used for the Faradaic efficiency measurements.

S19
The linear response of the setup was verified by injecting given amounts of molecular hydrogen in the gas tight cell and subsequently measuring the hydrogen GC peak area. Figure S12 clearly shows the linear relation between the hydrogen injected amount and the measured peak area. The solid line represents the best fit to the experimental points. For the Faradaic efficiency (FE) determination, the cell temperature was stabilized at 30°C and the electrolysis was initiated simultaneously to the GC acquisition. A potential of -0.6 V vs. SHE was applied for 1.5 h. The current passing through the electrode was continuously monitored and GC spectra were recorded each 5 min. At the end of each experiment, a 100 µL injection of molecular hydrogen was performed to calibrate the response of the GC. The FE was then calculated by comparing the number of hydrogen moles produced (GC peak area) with the expected amount of hydrogen as determined by the electric current timeintegration and applying the Faraday's laws of electrolysis. The measurement was repeated 3 times and the FE was found to be 98.5 ± 3.6 %. S20 S13 Long-term Faradaic efficiency of the CpI-nanoITO bioelectrode Figure 2b in the main article confirms that the current density for H 2 production remains stable over 5 days. During this revision stage of publishing this work, the Faradaic efficiency of the CpI-nanoITO bioelectrode was also confirmed over a 5-day period of continuous potentiostatic operation. The previously employed gas-tight cell S1 used in Figure 2b broke and a secondary gas-tight cell was employed. These experiments revealed the importance of efficient agitation of the electrochemical cell during continuous H 2 production. Figure S13a reports the evolution of the current density recorded over 5 days of continuous potentiostatic operation, using the cell which was no-longer gas-tight (broken), precluding the determination of Faradaic efficiency. Figure S13 reports the evolution of the current density (also recorded over 5 days) and Faradaic efficiency using a gas-tight cell in which good agitation conditions could not be achieved. It is important to note that the current density continuously decreased over 5 days. We hypothesize that this could be due to poor mass transport in the vicinity of the nanoITO-CpI electrode, which could result in a relatively basic pH (in comparison with the bulk) and the potential deactivation of CpI. Nevertheless, the Faradaic efficiency for H 2 production was determined to be 102.9 ± 1.4% (n = 7 GC readings of the same samples headspace) after 5 days of continuous operation. Figure S13c (photo) shows the accumulation of gas bubbles (presumably H 2 ) in this cell, as well as the sub-parr cell design that ultimately impedes efficient agitation. We conclude that efficient agitation (for H + delivery and H 2 removal) is critical to the operation of this nanoITO-CpI bioelectrodes. Figure S13: (a) Amperometric j -t curve of a GCE-nanoITO-hydrogenase bioelectrode at -0.6 V vs. SHE over 120 hours of continuous operation. Despite efficient stirring, this cell was not gas-tight. (b) Amperometric j -t curve of a GCE-nanoITO-hydrogenase bioelectrode at -0.6 V vs. SHE over 120 hours of continuous operation. This cell was technically gastight, although efficient stirring could not be achieved. The right-hand y-axis reports the Faradaic efficiency of this experiment (for H2) production, as determined by manual GC-TCD injections at the indicated times. Single injections were performed with the exception of the final data point (7 injection repeats). (c) Photograph of the cell used in (b), where H 2 bubble accumulation can be seen. The narrow conical design of this cell impedes efficient agitation using a magnetic stirrer.

S14 Inverted rotating disk electrode (iRDE) setup
The performance of the nanoITO hydrogenase was evaluated using an inverted RDE ( Figure S14: Inverted RDE (iRDE) and electrochemical cell assembly.

S23
A video demonstrating the working condition of the iRDE setup has been included in the Supporting Information. Figure Figure S15: Cross-sectional representation of the iRDE setup. The H-type cell fitting on top of the instrument is connected to the counter electrode, reference electrode, feeding gas inlet and a gas chromatograph for gas product analysis through tight stationary sealed connections. POM and PTFE stand for polyoxymethylene and polytetrafluoroethylene, respectively.

S15 SDS-PAGE
The protein was analyzed for its purity and molecular weight by SDS-PAGE analysis.
mPAGE™ 10% Bis-Tris Precast Gels were used. The protein was denatured using 0.1 M dithiothreitol (DTT) and by heating the mixture in 4× loading dye at 70°C for 10 min before loading onto the gel. The gel was run in 1× SDS-PAGE Running Buffer at 180 V as the final voltage. The gel was stained with 1× Biotium One-Step Blue ® Protein Gel Stain, and then imaged on Vilber Fusion FX imager.